We have determined the three-dimensional structures of the wild-type Sindbis virus and two mutants, each of which retains the E3 sequence within PE2. Using difference imaging between these mutants and the wild-type, we have located the 64 amino acid sequence corresponding to E3 in the mutant spike complex. In the wild-type the spike is composed of an E1-E2 heterotrimer. The E3 sequence was found to protrude midway between the center of the spike complex and the tips. Based on these results, we propose a model for the organization of the functional domains of the spike proteins in the structure of wild-type Sindbis virus. In this model, the E1 domains form the central portion of the spike complex; the tips are formed by E2 domains that flare out from the center of the spike. The structural similarity between these Sindbis mutants and Ross River virus suggests that E3 may also be present in the latter, which is also a member of the Alphaviridae genus.